The objective of this proposal is to develop 3D electrophoretic NMR spectroscopy and to identify protein folding intermediates with the technique. At present, NMR methods are capable of characterizing structures of pure proteins, strongly bound protein complexes, and the limited protein mixtures that can be resolved by chemical shifts. However, these methods cannot resolve resonances of multiple protein mixtures with overlapping NMR signals. The proposed novel methods will extend NMR technology for structural determination to meet these challenges. Electrophoretic mobility-ordered, homonuclear-ordered coherence correlation spectroscopy will be developed. By applying a pulsed electric field and magnetic field gradients, the investigators will introduce a dimension of electrophoretic mobility to the homonuclear-correlated NMR spectroscopy. These added dimensions will allow two-dimensional (2D) NMR spectra of different proteins to be sorted according to their electrophoretic mobilities. The plans are to accomplish the following specific aims: 1) develop 3D electrophoretic mobility-ordered correlation spectroscopy (COSY), double-quantum filtered COSY (DQF-COSY), total correlation spectroscopy (TOCSY), and nuclear overhauser enhancement spectroscopy (NOESY) techniques, demonstrating these methods with well-characterized zinc finger peptides, isozyme, and ubiquitin; 2) identify protein folding intermediates and simultaneously observe NMR spectra of co-existing conformations using alpha-lactalbumin (aLA) molten globule as an example. If proven, the development and application of the proposed novel NMR methods may have significant impact to many fields including: protein-protein or protein-DNA interactions, protein folding, enzyme kinetics, and structure-guided protein drug design.